Human milk compositions and methods of making and using same

ABSTRACT

The disclosure features human milk fortifier compositions, standardized human milk, and methods of making and using same. In one embodiment, a pasteurized human milk composition includes a human protein constituent of about 35-85 mg/mL; a human fat constituent of about 60-110 mg/mL; and a human carbohydrate constituent of about 60-140 mg/mL.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional application 60/841,371filed Aug. 30, 2006; provisional application 60/867,748, filed Nov. 29,2006; and PCT application PCT/US07/19234, filed Aug. 30, 2007. Thecontents of these applications are incorporated herein by reference intheir entireties.

TECHNICAL FIELD

The disclosure relates to human milk compositions, e.g., human milkfortifiers and standardized human milk formulations, and methods ofmaking and using such compositions.

BACKGROUND

Human milk is generally the food of choice for preterm and term infantsbecause of its nutritional composition and immunologic benefits. Thesource of human milk can be, e.g., a donor or the infant's mother. Thenutritional value of raw or conventionally-processed donor milk,however, varies and, in most instances, is not sufficient to meet theneeds of preterm infants. In addition, a possibility of bacterial, viraland other contamination of raw donor milk exists. For these and otherreasons, use of milk from the infant's own mother has become thepreferred nutritional approach in the modern neonatal intensive careunits (NICUs). Even the mother's own milk, however, is not nutritionallysufficient for the premature infant. It is often desirable to feedpreterm infants milk that is fortified with various supplements, e.g.,proteins, added energy (calories) and/or minerals.

SUMMARY

This disclosure features human milk compositions, e.g., pasteurizedhuman milk compositions, and methods of making and using suchcompositions. The compositions include human milk fortifiers (e.g.,PROLACTPLUS™ Human Milk Fortifiers, e.g., PROLACT+4™, PROLACT+6™,PROLACT+8™, and/or PROLACT+10™), which are produced from human milk andcontain various concentrations of nutritional components, e.g., protein,fat, carbohydrates, vitamins, and/or minerals. These fortifiers can beadded to the milk of a nursing mother to provide an optimal nutritionalcontent of the milk for, e.g., a preterm infant. Depending on thecontent of mother's own milk, various concentrations of the fortifierscan be added to mother's milk. For example, the protein concentration ofthe mother's milk can be increased with the use of the fortifier. Asmentioned above, the fortifiers of the present disclosure are generatedfrom human milk and, therefore, provide infants with human-derivednutrients.

The disclosure also features standardized human milk formulations(exemplified by PROLACT20™, NEO20™, and/or PROLACT24), which areproduced from human milk. Methods of making and using such compositionsare also described herein. These standardized human milk formulationscan be used to feed, e.g., preterm infants, without mixing them withother fortifiers or milk. They provide a nutritional human-derivedformulation and can substitute for mother's milk. Human milkformulations can contain various caloric contents, for example,PROLACT24™ (a full-feed whole milk product) can contain about 24 Cal/ozor about 81 Cal/100 mL.

The methods featured herein are used to process large volumes of donormilk, e.g., about 75-2,000 liters/lot of starting material.

In one aspect, the disclosure features a pasteurized human milkcomposition that includes: a human protein constituent of about 35-85mg/mL; a human fat constituent of about 60-110 mg/mL; and a humancarbohydrate constituent of about 60-140 mg/mL. The carbohydrateconstituent can include lactose. The composition can further include IgAand/or one or more constituents selected from the group consisting of:calcium, chloride, copper, iron, magnesium, manganese, phosphorus,potassium, sodium, and zinc. In one embodiment, the composition can bemixed with raw human milk to provide a nutritional composition, whereinthe raw human milk comprises about 80%, about 70%, about 60%, or about50% of the nutritional composition.

Embodiments can include one or more of the following features.

In one embodiment, the composition can include the protein constituentof about 55-65 mg/mL; the fat constituent of about 85-95 mg/mL; and thecarbohydrate constituent of about 70-120 mg/mL. The carbohydrateconstituent can include lactose. The composition can further include IgAand/or one or more constituents selected from the group consisting of:calcium (e.g., at a concentration of about 4.0-5.5 mg/mL or at 2.00-2.90mg/mL); chloride (e.g., at a concentration of about 0.35-0.95 mg/mL orat about 0.175-0.475 mg/mL); copper (e.g., at a concentration of about0.0005-0.0021 mg/mL or at about 0.00025-0.001 mg/mL); iron (e.g., at aconcentration of about 0.001-0.007 mg/mL or at about 0.0005-0.0025mg/mL); magnesium (e.g., at a concentration of about 0.180-0.292 mg/mLor at about 0.090-0.170 mg/mL); manganese (e.g., at a concentration ofabout 0.010-0.092 mcg/mL or at about 0.005-0.046 mcg/mL;); phosphorus(e.g., at a concentration of about 2.00-3.05 mg/mL or at about 1.00-2.90mg/mL, e.g., at about 1.00-1.50 mg/mL); potassium (e.g., at aconcentration of about 1.90-2.18 mg/mL or at about 0.95-1.41 mg/mL);sodium (e.g., at a concentration of about 0.75-0.96 mg/mL or at about0.375-0.608 mg/mL); and zinc (e.g., at a concentration of about0.0200-0.0369 mg/mL or at about 0.010-0.0198 mg/mL). In one embodiment,the composition can be mixed with raw human milk to provide anutritional composition, wherein the raw human milk comprises about 80%,about 70%, about 60%, or about 50% of the nutritional composition.

In another aspect, the disclosure features a pasteurized human milkcomposition that includes: a human protein constituent of about 11-20mg/mL, e.g., about 11-13 mg/mL; a human fat constituent of about 35-55mg/mL; and a human carbohydrate constituent of about 70-120 mg/mL, e.g.,about 80-105 mg/mL. The carbohydrate constituent can include lactose.The caloric content of the composition can be about 0.64 to about 1.10Cal/mL.

Embodiments can include one or more of the following features.

In one embodiment, the pasteurized human milk composition can furtherinclude one or more of the following components: calcium (e.g., at aconcentration of about 0.40-1.50 mg/mL); chloride (e.g., at aconcentration of about 0.30-0.80 mg/mL); copper (e.g., at aconcentration of about 0.0005-0.0021 mg/mL); iron (e.g., at aconcentration of about 0.001-0.005 mg/mL); magnesium (e.g., at aconcentration of about 0.03-0.13 mg/mL); manganese (e.g., at aconcentration of about 0.01-0.092 mcg/mL); phosphorus (e.g., at aconcentration of about 0.15-0.631 mg/mL); potassium (e.g., at aconcentration of about 0.60-1.20 mg/mL); sodium (e.g., at aconcentration of about 0.20-0.60 mg/mL); and/or zinc (e.g., at aconcentration of about 0.0025-0.0120 mg/mL).

The disclosure also features method of making various human milkcompositions.

In one aspect, the disclosure features a method for obtaining apasteurized human milk composition. The method includes: (a) geneticallyscreening human milk for one or more viruses; (b) filtering the milk;(c) heat-treating the milk, e.g., at about 63° C. or greater for about30 minutes; (d) separating the milk into cream and skim; (e) adding aportion of the cream to the skim; and (f) pasteurizing.

Embodiments include one or more of the following features.

In one embodiment, the method can further include filtering the skimthrough filters after step (d), e.g., to filter the water out of theskim. After filtering the skim after step (d), the filters used in thefiltering can be washed to obtain a post wash solution. The post washsolution can be added to the skim.

The genetic screening in step (a) can be polymerase chain reactionand/or can include screening for one or more viruses, e.g., HIV-1, HBV,and/or HCV. The milk can be filtered through an about 200 micron screenin step (b). The method can further include running cream, e.g., about30-50% of cream, through a separator following step (d). The compositionof post wash and skim can include about 7.0-7.2% of protein.

The method can further include carrying out mineral analysis of theportion of the composition obtained after step (e). The method can alsoinclude adding to the composition obtained after step (e) one or moreminerals selected from the group consisting of: calcium, chloride,copper, iron, magnesium, manganese, phosphorus, potassium, sodium, andzinc. Adding of the one or more minerals can include heating thecomposition.

The method can also include cooling the composition after step (f),carrying out biological testing of a portion of the composition afterstep (f), and/or carrying out nutritional testing of a portion of thecomposition after step (f).

The human milk of step (a) can be pooled human milk. The methodsfeatured herein can be carried out with large volumes of the startingmaterial, e.g., human milk, e.g., pooled human milk. The volumes can bein the range of about 75-2,000 liters/lot of starting material.

The composition obtained after step (f) can include about 8.5-9.5% fat,about 6.3-7.0% protein, and about 8.0-10.5% lactose.

In another aspect, the disclosure features a method for obtaining apasteurized human milk composition. The method includes: (a) geneticallyscreening human milk for one or more viruses; (b) filtering the milk;(c) adding cream; and (d) pasteurizing.

Embodiments can include one or more of the following features.

In one embodiment, the genetic screening in step (a) can be polymerasechain reaction. The genetic screening can include screening for one ormore viruses, e.g., HIV-1, HBV, and/or HCV.

The milk can be filtered through an about 200 micron screen in step (b).The method can further include ultra filtering the whole milk after step(b) through filters. The filters used during ultra filtering can be postwashed.

The composition can be cooled after step (d). Biological and/ornutritional testing of the composition can be carried out after step(d).

Human milk of step (a) can be pooled human milk. The methods featuredherein can be carried out with large volumes of the starting material,e.g., human milk, e.g., pooled human milk. The volumes can be in therange of about 75-2,000 liters/lot of starting material.

The method can also include adding to the composition obtained afterstep (c) one or more minerals selected from the group consisting of:calcium, chloride, copper, iron, magnesium, manganese, phosphorus,potassium, sodium, and zinc. In one embodiment, the composition obtainedafter step (d) can include about 11-20 mg/mL protein, about 35-55 mg/mLfat, and about 70-120 mg/mL carbohydrates.

In another aspect, the disclosure features a kit that includes thepasteurized human milk compositions featured herein (e.g., a fortifier)and a graduated container (e.g., a bottle, a syringe, and a can) formixing the featured compositions with raw human milk.

In yet another aspect, the disclosure features a method of obtaining anutritional milk composition. The method includes adding the pasteurizedhuman milk compositions featured herein (e.g., fortifiers) to raw humanmilk, thereby increasing the nutritional concentration of the raw humanmilk. The caloric composition of the raw human milk can be increased byabout 2-10 Cal/oz.

In another aspect, the disclosure features a method of providingsupplemental nutrients to a premature human infant, the methodcomprising adding the compositions (fortifiers) featured herein to rawhuman milk to obtain a mixture and administering the mixture to thepremature infant.

The terms “premature”, “preterm” and “low-birth-weight (LBW)” infantsare used interchangeably and refer to infants born less than 37 weeksgestational age and/or with birth weights less than 2500 gm.

By “whole milk” is meant milk from which no fat has been removed.

By “bioburden” is meant microbiological contaminants and pathogens(generally living) that can be present in milk, e.g., viruses, bacteria,mold, fungus and the like.

All patents, patent applications, and references cited herein areincorporated in their entireties by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart of an embodiment of a method of making a human milkfortifier.

FIG. 2 is a chart of an embodiment of a method of making standardizedhuman milk.

DETAILED DESCRIPTION

This disclosure features human milk compositions, e.g., pasteurizedhuman milk compositions, and methods of making and using suchcompositions. The compositions include human milk fortifiers (e.g.,PROLACTPLUS™ Human Milk Fortifiers, e.g., PROLACT+4™, PROLACT+6™,PROLACT+8™, and/or PROLACT+10™), which are produced from human milk andcontain various concentrations of nutritional components, e.g.,vitamins, and/or minerals. These fortifiers can be added to the milk ofa nursing mother to provide an optimal nutritional content of the milkfor, e.g., a preterm infant. Depending on the content of mother's ownmilk, various concentrations of the fortifiers can be added to mother'smilk. For example, the protein concentration and/or caloric content ofthe mother's milk can be increased with the use of the fortifier. In oneembodiment, the featured fortifiers can deliver from about 3.3 to about5.5 g of protein/kg/day to the infant receiving a full feed of 150 mLand/or 3.2 to 4.1 grams of protein per 120 Cal/kg/day.

The disclosure also features standardized human milk formulations(exemplified by PROLACT20™, NEO20™, and/or PROLACT24™), which areproduced from human milk. Methods of making and using such compositionsare also described. These standardized human milk formulations can beused to feed, e.g., preterm infants, without mixing them with otherfortifiers or milk. Thus, the compositions provide an infant with ahuman-derived nutritional formulation that can substitute for mother'smilk. Human milk formulations can contain various caloric contents, forexample, PROLACT24 (a full feed whole milk formulation) can containabout 24 Cal/oz or about 81 Cal/100 mL.

The compositions of the present disclosure are generated from humandonor milk, e.g., pooled milk, which undergoes rigorous geneticscreening, processing (e.g., to concentrate nutrients in the fortifiercompositions, and/or to reduce bioburden), and pasteurization. The milkcan be supplemented with various minerals and/or vitamins. Thus, thedisclosure also features methods of obtaining and processing milk fromhuman donors.

The methods of the present disclosure can be used to process largevolumes of donor milk, e.g., about 75-2,000 liters/lot of startingmaterial.

Nutritional Requirements of Premature Infants

There are many factors that can affect the clinical outcome of anewborn, e.g., prematurely born, infant. Preterm infants have vulnerableimmune systems, immature digestive systems, and increased total caloricand specific nutrient needs (when generally compared with term infants).Thus, nutrition provided to such infants is an important factor in theirgrowth and development. Human milk has long been recognized as the idealfood for preterm and term infants because of its nutritional compositionand immunologic benefits. Not every mother, however, can or willbreastfeed her baby (or use a breast pump and store her milk). Forexample, mothers who have certain diseases, e.g., active tuberculosis,or are being administered radioisotopes, antimetabolites, orchemotherapy may not breast feed their infants. In addition, mother'sown milk may not contain sufficient nutritional content to sustain apreterm infant. Use of donor milk can also be problematic, as such milkmay not contain adequate nutrition for a preterm infant.

The present disclosure features human milk compositions and methods ofmaking and using such compositions for feeding and/or increasing thenutritional value of milk fed to infants, e.g., premature infants. Thefortifiers described herein (e.g., PROLACTPLUS™) can deliver a highlevel of, e.g., protein and/or calories to the milk and therefore to theinfant. The standardized human milk formulations (e.g., PROLACT20™ andNEO20™) can be used instead of mother's own milk. The compositions canbe supplemented with various vitamins and/or minerals. The compositionscan also contain IgA (e.g., secretory IgA) and various componentsdescribed herein.

Obtaining Human Milk from Qualified and Selected Donors

The compositions of the present disclosure are generated from donatedhuman milk. Various techniques are used to identify and qualify suitabledonors. A potential donor must obtain a release from her physician andher child's pediatrician as part of the qualification process. Thishelps to insure, inter alia, that the donor is not chronically ill andthat her child will not suffer as a result of the donation(s). Methodsand systems for qualifying and monitoring milk collection anddistribution are described, e.g., in U.S. patent application Ser. No.11/526,127 (U.S. 2007/0098863), which is incorporated herein byreference in its entirety.

Generally, the donor screening process includes both interviews andbiological sample processing. Any blood sample found positive for, e.g.,viral contamination, on screening removes the donor from thequalification process.

Once a donor qualifies and begins sending milk, milk from each of hershipments is tested for, e.g., B. cereus and drugs of abuse (e.g.,cocaine, opiates, methamphetamines, benzodiazepine, amphetamines, andTHC). Any positive finding results in the deferral of the donor anddestruction of all previously-collected milk.

Donors may be periodically requalified. For example, a donor is requiredto undergo screening by the protocol used in their initial qualificationevery four months, if the donor wishes to continue to donate. A donorwho does not requalify or fails qualification is deferred until suchtime as they do, or permanently deferred if warranted by the results ofrequalification screening. In the event of the latter situation, allremaining milk provided by that donor is removed from inventory anddestroyed.

A qualified donor may donate at a designated facility (e.g., a milk bankoffice) or, typically, expresses milk at home. The qualified donor canbe provided with supplies by a milk bank or directly from a milkprocessor (the milk bank and processor may be the same or differententities) to take home. The supplies will typically comprise a computerreadable code (e.g., a barcode-label) on containers and may furtherinclude a breast pump. The donor can then pump and freeze the milk athome at a temperature of about −20° C. or colder. The donor milk isaccepted, provided that the donor is a qualified donor; if such resultsare satisfactory, an appointment is made for the donor to drop off themilk at the center, or to have it collected from home. A donor can alsoship the milk directly to the milk bank center or milk processor ininsulated containers provided by the milk bank or milk processor. Themilk and container are examined for their condition and the barcodeinformation checked against the database. If satisfactory, the units areplaced in the donor milk center or processing center freezer (−20° C. orcolder) until ready for further testing and processing.

Because in some embodiments of the present methods the milk is expressedby the donor at her home and not collected at the milk banking facility,each donor's milk is sampled for genetic markers, e.g., DNA markers, toguarantee that the milk is truly from the registered donor. Such subjectidentification techniques are known in the art (see, e.g., InternationalApplication Serial No. PCT/US2006/36827, which is incorporated herein byreference in its entirety). The milk may be stored (e.g., at −20° C. orcolder) and quarantined until the test results are received. Throughoutthe above process, any non-complying milk specimens are discarded, andthe donor is disqualified. Access to all confidential information aboutthe donor, including blood test data, is carefully controlled and meetsHealth Insurance Portability and Accountability Act (HIPAA)requirements.

Processing Donated Human Milk

Collected donor human milk is processed to obtain, e.g., a human milkfortifier, and/or standardized human milk formulation.

Methods of Obtaining Human Milk Fortifiers

FIG. 1 is a chart showing an embodiment of generating a human milkfortifier (e.g., PROLACTPLUS™). A detailed embodiment is discussed inExample 1 below. As discussed above, donor milk is carefully analyzedfor both identification purposes and to avoid contamination. The donormilk is frozen and, when desired, thawed and pooled. It is then screened(step 1 of FIG. 1), e.g., genetically screened, e.g., by polymerasechain reaction (PCR). Genetic screening is done to identify anycontaminants, e.g., viral, e.g., HIV-1, HBV, and/or HCV. The milk thenundergoes filtering, e.g., through about a 200 micron filter (step 2),and heat treatment (step 3). For example, the composition can be treatedat about 63° C. or greater for about 30 minutes or more. In step 4, themilk is transferred to a separator, e.g., a centrifuge, to separate thecream from the skim. The skim can be transferred into a secondprocessing tank where it remains at about 2 to 8° C. until a filtrationstep (step 5).

Optionally, the cream separated from the skim in step 4, can undergoseparation again to yield more skim.

Following separation of cream and skim (step 4), a desired amount ofcream is added to the skim, and the composition undergoes furtherfiltration (step 5), e.g., ultrafiltration. This process concentratesthe nutrients in the skim milk by filtering out the water. The waterobtained during the concentration is referred to as the permeate.Filters used during the ultrafiltration can be postwashed and theresulting solution added to the skim to maximize the amount of nutrientsobtained, e.g., obtaining a protein concentration of about 7% to 7.2%.The skim is then blended with the cream (step 6) and samples taken foranalysis. At this point during the process, the composition generallycontains: about 8.5% to 9.5% of fat; about 6.3% to 7.0% of protein; andabout 8% to 10.5% of carbohydrates, e.g., lactose.

After the separation of cream and skim in step 4, the cream flows into aholding tank, e.g., a stainless steel container. The cream can beanalyzed for its caloric, protein and fat content. When the nutritionalcontent of cream is known, a portion of the cream can be added to theskim milk that has undergone filtration, e.g., ultrafiltration, (step 5)to achieve the caloric, protein and fat content required for thespecific product being made. Minerals can be added to the milk prior topasteurization.

At this point, in one embodiment, the processed composition can befrozen prior to the addition of minerals and thawed at a later point forfurther processing. Any extra cream that was not used can also bestored, e.g., frozen. Optionally, before the processed composition isfrozen, samples are taken for mineral analysis. Once the mineral contentof the processed milk is known, the composition can be thawed (if itwere frozen) and a desired amount of minerals can be added to achievetarget values.

After step 6 and/or the optional freezing and/or mineral addition, thecomposition undergoes pasteurization (step 7). For example, thecomposition can be placed in a process tank that is connected to thehigh-temperature, short-time (HTST) pasteurizer via platinum-curedsilastic tubing. After pasteurization, the milk can be collected into asecond process tank and cooled. Other methods of pasteurization known inthe art can be used. For example, in vat pasteurization the milk in thetank is heated to a minimum of 63° C. and held at that temperature for aminimum of thirty minutes. The air above the milk is steam heated to atleast three degrees Celsius above the milk temperature. In oneembodiment, the product temperature is about 66° C. or greater, the airtemperature above the product is about 69° C. or greater, and theproduct is pasteurized for about 30 minutes or longer. In anotherembodiment, both HTST and vat pasteurization are performed.

The resulting fortifier composition is generally processed aseptically.After cooling to about 2 to 8° C., the product is filled into containersof desired volumes, and various samples of the fortifier are taken fornutritional and bioburden analysis. The nutritional analysis ensuresproper content of the composition. A label that reflects the nutritionalanalysis is generated for each container. The bioburden analysis testsfor presence of contaminants, e.g., total aerobic count, B. cereus, E.coli, Coliform, Pseudomonas, Salmonella, Staphylococcus, yeast, and/ormold. Bioburden testing can be genetic testing. The product is packagedand shipped once the analysis is complete and desired results areobtained.

Methods of Obtaining Standardized Human Milk Formulations

FIG. 2 and Examples 3 and 4 below show some embodiments of the methodsfor obtaining standardized human milk formulations (e.g., PROLACT20™ andNEO20™)

As discussed above, donor milk is screened to ensure the identity of thedonors and reduce the possibility of contamination. Donor milk is pooledand further screened (step 1 of FIG. 2), e.g., genetically screened(e.g., by PCR). The screening can identify, e.g., viruses, e.g., HIV-1,HBV, and/or HCV. Milk that tests positive is discarded. After thescreening, the composition undergoes filtering (step 2). The milk isfiltered through about a 200 micron screen and then ultrafiltered.During ultrafiltration, water is filtered out of the milk (and isreferred to as permeate) and the filters are postwashed using thepermeate. Post wash solution is added to the milk to recover any lostprotein and increase the concentration of the protein to, e.g., about1.2% to about 1.5%. Referring to FIG. 2, cream from another lot (e.g.,excess cream from a previous fortifier lot) is added in step 3 toincrease the caloric content. At this stage of the process, thecomposition generally contains: about 3.5% to 5.5% of fat; about 1.1% to1.3% of protein; and about 8% to 10.5% of carbohydrates, e.g., lactose.

At this stage, the composition can be frozen and thawed out for furtherprocessing later.

Optionally, if the human milk formulation is to be fortified withminerals, a mineral analysis of the composition is carried out afterstep 3. Once the mineral content is known, a desired amount of mineralscan be added to achieve target values.

In step 4, the composition is pasteurized. Pasteurization methods areknown in the art. For example, the product can be pasteurized in a tankthat is jacketed. Hot glycol can be use to heat up the tank. The producttemperature can be about 63° C. or greater and the air temperature abovethe product about 66° C. or greater. The product is pasteurized for aminimum of about 30 minutes. Other pasteurizing techniques are known inthe art.

After cooling to about 2 to 8° C., the product is filled into containersof desired volumes and various samples of the human milk formulation aretaken for nutritional and bioburden analysis. The nutritional analysisensures proper content of the composition. A label generated for eachcontainer reflects the nutritional analysis. The bioburden analysistests for presence of contaminants, e.g., total aerobic count, B.cereus, E. coli, Coliform, Pseudomonas, Salmonella, Staphylococcus,yeast, and/or mold. The product is packaged and shipped once theanalysis is complete and desired results are achieved.

Effects of the Featured Methods on Components and Bioburden of HumanMilk

Human milk comprises about 100,000 different molecularentities—proteins, lipids, carbohydrates, vitamins, and trace minerals.Some specific components include the monomeric immunoglobulin A (IgA)and the dimeric secretory IgA (s[IgA]₂), lysozyme, and lactoferrin.Accordingly, the methods featured herein (which include pasteurization),while helping to ensure the safety of the milk product, should alsopreserve adequate quantities of those components, which are potentiallyheat labile. As described in Example 5 below and Table 3, pasteurizationmethods featured herein can preserve a large amount of IgA, lysozyme,and Vitamin B6 in the processed milk. As described in Example 5 belowand Table 2, the featured methods are able to reduce any bioburden ofhuman milk.

Human Milk Compositions

The compositions featured herein contain various amounts of nutrients,e.g., protein, carbohydrates, fat, vitamins, and minerals, as well asother milk components, such as IgA and lysozyme. Human milk fortifiersprovide a concentrated amount of nutrients and calories that can beadded to, e.g., mother's milk. Standardized human milk formulations canbe supplemented with vitamins and/or minerals if desired and can be feddirectly to an infant, e.g., a premature infant. The methods ofgenerating these compositions are designed to optimize the amount ofnutrients and calories in the compositions. For example, thecompositions featured herein can deliver from about 3.3 to about 5.5grams of protein/kg/day to an infant receiving a full feeding of 150 mLper day and/or 3.2 to 4.1 grams of protein/kg/day to infants receiving120 Cal/kg/day.

Human Milk Fortifiers

The human milk fortifiers featured herein contain concentrated amountsof nutrients. In one embodiment, the fortifier can contain a humanprotein constituent of about 35-85 mg/mL; a human fat constituent ofabout 60-110 mg/mL; and a human carbohydrate constituent of about 60-140mg/mL. Total caloric content of the fortifier can be, e.g., about 0.92to about 1.89 Cal/mL. In particular, one embodiment can include theprotein constituent of about 55-65 mg/mL; the fat constituent of about85-95 mg/mL; and the carbohydrate constituent of about 70-120 mg/mL.

The fortifier can be supplemented with minerals, if desired. In oneembodiment, the minerals can include: calcium concentration of about4.0-5.5 mg/mL; chloride concentration of about 0.35-0.95 mg/mL; copperconcentration of about 0.0005-0.0021 mg/mL; iron concentration of about0.001-0.700 mg/mL; magnesium concentration of about 0.180-0.292 mg/mL;manganese concentration of about 0.010-0.092 micrograms/mL; phosphorusconcentration of about 2.00-3.05 mg/mL; potassium concentration of about1.90-2.18 mg/mL; sodium concentration of about 0.75-0.96 mg/mL; and zincconcentration of about 0.0200-0.0396 mg/mL. In another embodiment, theminerals can include calcium concentration of about 2.00-2.9 mg/mL;chloride concentration of about 0.175-0.475 mg/mL; copper concentrationof about 0.00025-0.001 mg/mL; iron concentration of about 0.0005-0.0025mg/mL; magnesium concentration of about 0.090-0.170 mg/mL; manganeseconcentration of about 0.005-0.046 micrograms/mL; phosphorusconcentration of about 1.00-1.50 mg/mL; potassium concentration of about0.95-1.41 mg/mL; sodium concentration of about 0.375-0.608 mg/mL; andzinc concentration of about 0.010-0.0198 mg/mL.

An exemplary fortifier (e.g., PROLACT+4™ and PROLACT+6™) can include thefollowing components: human milk, calcium carbonate, potassiumphosphate, calcium phosphate, calcium glycerophosphate, calciumgluconate, sodium citrate, magnesium chloride, calcium chloride,magnesium phosphate, zinc sulphate, cupric sulfate, and manganesesulfate. An exemplary fortifier can have the following characteristicsper 100 mL: about 135-155 Cal; about 8.5-9.5 g of total fat; about 75-96mg of sodium; about 190-218 mg of potassium; about 7.0-12.0 g of totalcarbohydrates; about 5.5-10.0 g of sugars; about 5.5-6.5 g of protein;about 1000-5000 IU of Vitamin A; less than about 1 mg of Vitamin C;about 400-550 mg of calcium; about 0.1-0.5 mg of iron; about 200-305 mgof phosphorus; about 18-29.2 mg of magnesium; about 35-95 mg ofchloride; about 2.0-4.0 mg of zinc; about 0.05-0.21 mcg of copper; andless than about 9.2 mcg of manganese; with the osmolality of ˜343mOsm/Kg H₂O (when mixed with raw human milk at the ratio of about 80%raw milk to 20% fortifier).

Fortifiers with other constituents and constituents of differentconcentrations are encompassed by this disclosure.

The fortifiers featured herein can be mixed with mother's milk atvarious concentrations, depending on the nutritional content of themother's milk and the needs of the infant. For example, a mother's rawmilk can be tested to determine the raw milk's nutritional value.Typical raw milk comprises, on average, 1.1 g protein, 3.2 g fat, 7.7 gcarbohydrates (primarily lactose), and supplies about 64 kcal of energyper 100 mL. After testing, the mother's milk can be adjusted by adding afortifier composition discussed herein. For example, the fortifiers canadd from about 2 to about 10 Cal/oz and/or from 3.3 to 5.5g/protein/kg/day when considering a full feeding of 150 mL/day and/or3.2 to 4.1 grams of protein/kg/day to infants receiving 120 Cal/kg/day.If a lower dose of the fortifier and the nutrients is needed, themixture can include about 20% of the fortifier and about 80% of rawhuman milk (adding about 4 Cal/oz) (e.g., the mixture with PROLACT+4™).If a higher dose of the nutrients is needed, the mixture can includeabout 50% of raw human milk and about 50% of the fortifier (e.g., themixture with PROLACT+10™. Other ratios of the mixtures are encompassedby this disclosure, including (but not limited to): about 30% fortifierwith about 70% raw human milk (e.g., the mixture with PROLACT+6™) andabout 40% of fortifier with about 60% of raw human milk (e.g., themixture with PROLACT+8™). Example 2 below and Table 2 show someembodiments of the mixtures and their nutritional information.

Standardized Human Milk Formulations

The standardized human milk formulations featured herein can be used inlieu of mother's own milk to feed the infants, e.g., premature infants.They include various nutritional components for infant growth anddevelopment.

In one embodiment, the standardized human milk formulation can include:a human protein constituent of about 11-20 mg/mL; a human fatconstituent of about 35-55 mg/mL; and a human carbohydrate constituentof about 70-120 mg/mL. In a particular embodiment, the formulation cancontain: a human protein constituent of about 11-13 mg/mL; a human fatconstituent of about 35-55 mg/mL; and a human carbohydrate constituentof about 80-105 mg/mL. The total caloric content of the formulations canbe, e.g., from about 0.68 Cal/mL to about 0.96 Cal/mL.

The milk formulation can be supplemented with vitamins and/or minerals.In one embodiment, the composition can include: calcium concentration ofabout 0.40-1.50 mg/mL; chloride concentration of about 0.30-0.80 mg/mL;copper concentration of about 0.0005-0.0021 mg/mL; iron concentration ofabout 0.001-0.005 mg/mL; magnesium concentration of about 0.03-0.13mg/mL; manganese concentration of about 0.01-0.092 mg/mL; phosphorusconcentration of about 0.15-0.631 mg/mL (e.g., about 0.15-0.60 mg/mL);potassium concentration of about 0.60-1.20 mg/mL; sodium concentrationof about 0.20-0.60 mg/mL; and zinc concentration of about 0.0025-0.0120mg/mL.

The human milk formulations can contain various caloric content, e.g.,67 Kcal/dL (20 Calorie per ounce), and 81 Kcal/dL (24 Calorie perounce). An exemplary human milk formulation (e.g., PROLACT24N caninclude the following constituents: human milk, calciumglycerophosphate, potassium citrate, calcium gluconate, calciumcarbonate, magnesium phosphate, sodium chloride, sodium citrate, zincsulfate, cupric sulfate, and manganese sulfate. This exemplarycomposition can have the following characteristics per 100 ml: about 81Cal; about 4.4 g of total fat; about 20.3 mg of sodium; about 60.3 mg ofpotassium; about 8 g total carbohydrates; about 5-9 g of sugars; about2.3 g of protein; about 180-250 IU of Vitamin A; less than about 1.0 mgof Vitamin C; about 40.0-150.0 mg of calcium; about 100-150 mcg of iron;about 15-50 mg of phosphorus; about 3-10 mg of magnesium; about 25-75.0mg of chloride; about 1.2 mcg of zinc; about 140-190 mcg of copper; lessthan about 60.2 mcg of manganese; and Osmolarity of about 322 mOsm/KgH₂O. Milk formulations with other constituents and constituents ofdifferent concentrations are encompassed by this disclosure.

Specific Components of the Featured Compositions

One component of the milk compositions and fortifiers featured herein isprotein. In the body, protein is needed for growth, synthesis of enzymesand hormones, and replacement of protein lost from the skin, urine andfeces. These metabolic processes determine the need for both the totalamount of protein in a feeding and the relative amounts of specificamino acids. The adequacy of the amount and type of protein in a feedingfor infants is determined by measuring growth, nitrogen absorption andretention, plasma amino acids, certain blood analytes and metabolicresponses. Some proteins present in the featured compositions beneficialfor other than purely nutritional reasons include human IgA, lysozyme,and lactoferrin.

Another constituent of the milk compositions described herein is fat.Fat is generally a source of energy for LBW infants, not only because ofits high caloric density but also because of its low osmotic activity insolution. Thus, optionally, the milk compositions of the invention canbe supplemented with fat constituents. Such heterologous fatconstituents include specific fatty acids such as docosahexaenoic acid(DHA) and arachidonic acid.

Vitamins and minerals are important to proper nutrition and developmentof an infant. A premature infant or LBW infant requires electrolytes,e.g., sodium, potassium and chloride for growth and for acid-basebalance. Sufficient intakes of these electrolytes are also needed forreplacement of losses in the urine and stool and from the skin. Calcium,phosphorus and magnesium are needed for proper bone mineralization andgrowth.

Trace minerals are associated with cell division, immune function andgrowth. Consequently, sufficient amounts of trace minerals are neededfor infant growth and development. Some trace minerals that areimportant include, e.g., copper, magnesium and iron (which is important,e.g., for the synthesis of hemoglobin, myoglobin and iron-containingenzymes). Zinc is needed, e.g., for growth, for the activity of numerousenzymes, and for DNA, RNA and protein synthesis. Copper is necessaryfor, e.g., the activity of several important enzymes. Manganese isneeded, e.g., for the development of bone and cartilage and is importantin the synthesis of polysaccharides and glyoproteins. Accordingly, thehuman milk formulations and fortifier compositions of the invention canbe supplemented with vitamins and minerals as described herein.

Vitamin A is a fat-soluble vitamin essential for, e.g., growth, celldifferentiation, vision and proper functioning of the immune system.Vitamin D is important, e.g., for absorption of calcium and to a lesserextent, phosphorus, and for the development of bone. Vitamin E(tocopherol) prevents peroxidation of polyunsaturated fatty acids in thecell, thus preventing tissue damage. Folic acid plays a role in, e.g.,amino acid and nucleotide metabolism. Serum folate concentrations havebeen shown to fall below normal after 2 weeks of age in LBW infants withlow folic acid intakes. Additionally, several B vitamins are present atlow concentrations in preterm milk.

As described above, the variability of human milk vitamin and mineralconcentrations and the increased needs of the preterm infant oftenrequire some fortification to insure that a developing infant isreceiving adequate amounts of vitamins and minerals. Examples ofvitamins and minerals that can be added to the human milk compositionsfeatured herein include: vitamin A, vitamin B1, vitamin B2, vitamin B6,vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin, folicacid, pantothenic acid, niacin, m-inositol, calcium, phosphorus,magnesium, zinc, manganese, copper, sodium, potassium, chloride, ironand selenium. The compositions can also be supplemented with: chromium,molybdenum, iodine, taurine, carnitine and choline may also requiresupplementation.

The osmolality of human milk fortifiers and standardized milkformulations featured herein can affect adsorption, absorption, anddigestion of the compositions. High osmolality, e.g., above about 400mOsm/Kg H₂O, has been associated with increased rates of necrotizingenterocolitis (NEC), a gastrointestinal disease that affects neonates(see, e.g., Srinivasan et al., Arch. Dis. Child Fetal Neonatal Ed.89:514-17, 2004). The osmolality of the human milk composition andfortifier (once mixed with raw milk) of the disclosure is typically lessthan about 400 mOsm/Kg H₂O. Typically the osmolality is from about 310mOsm/Kg of water to about 380 mOsm/Kg of water. The osmolality can beadjusted by methods known in the art.

Kits

The present disclosure also features kits that include the human milkfortifiers described herein and containers for mixing the fortifierswith raw human milk. The containers can include bottles, e.g., graduatedbottles to assist in proper dilution, syringes, cans, and othercontainers known in the art.

Processing of the Featured Compositions in NICU

Preparation of the fortifiers and standardized milk products in, e.g.,NICUs, is adjusted depending on the needs of the patients and thepolicies of various hospitals. Thus, the amount of milk prepared, e.g.,with the fortifiers, will be determined on site.

The embodiments of the disclosure may, of course, be carried out inother ways than those set forth herein without departing from the spiritand scope of the disclosure. The embodiments are, therefore, to beconsidered in all respects as illustrative and not restrictive.

Example 1 Production and Compositions of PROLACTA™ Bioscience Human MilkFortifier (HMF)

The PROLACTA™ Bioscience Human Milk Fortifier (HMF) (PROLACTPLUS™) wasproduced using the following steps. Processing was done in a class100,000 clean room (ISO class 8), and filling was done in a class 10,000clean room (ISO class 7):

1. Donor milk was pooled.

2. A sample was taken for Polymerase Chain Reaction (PCR) for thefollowing viruses:

a. HIV-1

b. HBV—Hepatitis B

c. HCV—Hepatitis C

3. Donor milk was filtered through a 200 micron screen.

4. Donor milk was heat-treated at 63° C. or greater for 30 minutes.

5. Milk was separated into in skim milk and cream, which had thefollowing concentrations:

a. Skim: 0.3%-0.69% Fat, 0.9%-1.2% Protein, 6%-10% Lactose

b. Cream: 25%-46% Fat, 0.5%-2% Protein, 8-10% Lactose

6. The Cream can be, if desired, taken and run through the separator:

a. This step produces more skim milk, which increases the yield becausethe more skim is produced, the more HMF can be produced.

b. The product that comes out the non-skim side of the separator iscurrently considered waste.

7. Cream was added to skim for better ultra filtration duringconcentration step. The fat in the skim was from 55% to 65% of theprotein content.

8. Skim milk underwent ultra filtration. This step filtered water out ofthe skim milk. The water was referred to as permeate. The finalconcentration during this step was 7%-10% of protein.

9. Post wash of the filters using permeate was carried out to recoverany protein that was trapped on the filters during the ultra filtrationprocess. The post wash was added to the concentrated skim milk untilprotein concentration reached 7.0%-7.2%.

10. During the Bulk formulation, cream was added to the concentratedskim milk, after post wash was added. This step increased calories tothe correct target. At this point, the product was at:

-   -   a. Fat—8.5%-9.5%;    -   b. Protein—6.3%-7.0%; and    -   c. Lactose—8%-10.5%.

11. A sample of the Bulk was sent for mineral analysis. This analysis ofthe initial mineral content allowed for later determination of theamount of minerals that needed to be added to the product (see, e.g.,step 14).

12. The Bulk was frozen at −20° C. or colder.

13. When the mineral results were returned from the lab, the Bulk wasthawed.

14. Based on the mineral concentration in the starting material(determined in step 11 above), it was calculated how much more of theminerals needed to be added.

The final targets are set out below. PROLACT+4™, PROLACT+6™, PROLACT+8™,and PROLACT+10™ fortifiers are used in mixtures with human milk and arefurther discussed in Example 2 below.

a. PROLACT+4™ and PROLACT+6™

-   -   i. Calcium—400.0 mg/dL to 550.0 mg/dL    -   ii. Chloride—35 mg/dL to 95.0 mg/dL    -   iii. Copper—0.05 mg/dL to 0.21 mg/dL    -   iv. Iron—0.1 mg/dL to 0.7 mg/dL    -   v. Magnesium—18.0 mg/dL to 29.2 mg/dL    -   vi. Manganese—1.0 micrograms/dL to 9.2 micrograms/dL    -   vii. Phosphorus—200.0 mg/dL to 305.0 mg/dL    -   viii. Potassium—190.0 mg/dL to 218.0 mg/dL    -   ix. Sodium—75 mg/dL to 96 mg/dL    -   x. Zinc—2.0 mg/dL to 3.96 mg/dL

b. PROLACT+8™ and PROLACT+10™

-   -   i. Calcium—200.0 mg/dL to 290 mg/dL    -   ii. Chloride—17.5 mg/dL to 47.5 mg/dL    -   iii. Copper—0.025 mg/dL to 0.1 mg/dL    -   iv. Iron—0.05 mg/dL to 0.25 mg/dL    -   v. Magnesium—9.0 mg/dL to 14.6 mg/dL    -   vi. Manganese—0.5 micrograms/dL to 4.6 micrograms/dL    -   vii. Phosphorus—100.0 mg/dL to 150.0 mg/dL    -   viii. Potassium—95.0 mg/dL to 141.0 mg/dL    -   ix. Sodium—37.5 mg/dL to 60.8 mg/dL    -   x. Zinc—1.0 mg/dL to 1.98 mg/dL

c. PROLACT+6™ will also be made with the following formulation:

-   -   i. Calcium—250.0 mg/dL to 425.0 mg/dL    -   ii. Chloride—15 mg/dL to 75.0 mg/dL    -   iii. Copper—0.05 mg/dL to 0.21 mg/dL    -   iv. Iron—0.1 mg/dL to 0.5 mg/dL    -   v. Magnesium—10.0 mg/dL to 25.0 mg/dL    -   vi. Manganese—1.0 micrograms/dL to 9.2 micrograms/dL    -   vii. Phosphorus—125.0 mg/dL to 225.0 mg/dL    -   viii. Potassium—60.0 mg/dL to 105.0 mg/dL    -   ix. Sodium—50.0 mg/dL to 90 mg/dL    -   x. Zinc—2.0 mg/dL to 3.96 mg/dL

15. The Final Bulk was heated to 50° C.-55° C. and the minerals weremixed into the Final Bulk.

16. After the minerals have been mixed, the product was pasteurized in atank. The tank was jacketed and hot glycol was used to heat up the tank.The following parameters were followed:

a. The product temperature was 66° C. or greater;

b. The air temperature above the product was 69° C. or greater; and

c. The product was pasteurized for a minimum of 30 minutes.

17. The product temperature was brought down to 2° C.-8° C. using coldglycol in the jacketed tank.

18. The product was filled into bottles using a Watson-Marlow fillingmachine in the class 10,000 clean room. The fill sizes were as follows:

a. PROLACT+4™ 10 mL

b. PROLACT+4™ 20 mL

c. PROLACT+4™ 100 mL

d. PROLACT+6™ 15 mL

e. PROLACT+6™ 30 mL

f. PROLACT+8™ 20 mL

g. PROLACT+8™ 40 mL

h. PROLACT+10™ 25 mL

i. PROLACT+10™ 50 mL.

19. When the fill was finished, the bottles were used as follows:

a. BioBurden samples—2 bottles from the beginning, middle and end of thefill run, for a total of 6 bottles were taken for tests;

b. Nutritional samples—1 bottle from the beginning, middle, and end ofthe fill run, for a total of 3 bottles were taken for tests; and

c. Retention samples—120 mL of product was retained.

20. The analysis was done on the BioBurden samples for the following:

a. Total Aerobic Count—<100 Colony Forming Unit/mL;

b. Bacillus cereus—<10 Colony Forming Unit/mL;

c. E. coli—<1 Colony Forming Unit/mL;

d. Coliform—<1 Colony Forming Unit/mL;

e. Pseudomonas—<1 Colony Forming Unit/mL;

f. Salmonella—<1 Colony Forming Unit/mL;

g. Staphylococcus—<1 Colony Forming Unit/mL;

h. Yeast—<100 Colony Forming Unit/mL; and

i. Mold—<100 Colony Forming Unit/mL.

21. The following nutritional analysis was conducted.

a. PROLACT+4™ or PROLACT+6™

-   -   i. Total Calories—1.35 Cal/mL to 1.55 Cal/mL    -   ii. Protein—5.5 g/dL to 6.5 g/dL    -   iii. Fat—8.5 g/dL to 9.5 g/dL    -   iv. Lactose—7.0 g/dL to 12.0 g/dL    -   v. Calcium—400.0 mg/dL to 550.0 mg/dL    -   vi. Chloride—35 mg/dL to 95.0 mg/dL    -   vii. Copper—0.05 mg/dL to 0.21 mg/dL    -   viii. Iron—0.1 mg/dL to 0.7 mg/dL    -   ix. Magnesium—18.0 mg/dL to 29.2 mg/dL    -   x. Manganese—1.0 micrograms/dL to 9.2 micrograms/dL    -   xi. Phosphorus—200.0 mg/dL to 305.0 mg/dL    -   xii. Potassium—190.0 mg/dL to 218.0 mg/dL    -   xiii. Sodium—75 mg/dL to 96 mg/dL    -   xiv. Zinc—2.0 mg/dL to 3.96 mg/dL

b. PROLACT+8™ or PROLACT+10™

-   -   i. Total Calories—1.35 Cal/mL to 1.55 Cal/mL    -   ii. Protein—5.5 g/dL to 6.5 g/dL    -   iii. Fat—8.5 g/dL to 9.5 g/dL    -   iv. Lactose—7.0 g/dL to 12.0 g/dL    -   v. Calcium—200.0 mg/dL to 275 mg/dL    -   vi. Chloride—17.5 mg/dL to 47.5 mg/dL    -   vii. Copper—0.025 mg/dL to 0.1 mg/dL    -   viii. Iron—0.05 mg/dL to 0.25 mg/dL    -   ix. Magnesium—9.0 mg/dL to 14.6 mg/dL    -   x. Manganese—0.5 micrograms/dL to 4.6 micrograms/dL    -   xi. Phosphorus—100.0 mg/dL to 150.0 mg/dL    -   xii. Potassium—95.0 mg/dL to 141.0 mg/dL    -   xiii. Sodium—37.5 mg/dL to 60.8 mg/dL    -   xiv. Zinc—1.0 mg/dL to 1.98 mg/dL

c. PROLACT+6™ will also be made with the following formulation:

-   -   i. Total Calories—1.35 Cal/mL to 1.55 Cal/mL    -   ii. Protein—5.5 g/dL to 6.5 g/dL    -   iii. Fat—8.5 g/dL to 9.5 g/dL    -   iv. Lactose—7.0 g/dL to 12.0 g/dL    -   v. Calcium—250.0 mg/dL to 425.0 mg/dL    -   vi. Chloride—35 mg/dL to 75.0 mg/dL    -   vii. Copper—0.05 mg/dL to 0.21 mg/dL    -   viii. Iron—0.1 mg/dL to 0.5 mg/dL    -   ix. Magnesium—10.0 mg/dL to 25.0 mg/dL    -   x. Manganese—1.0 micrograms/dL to 9.2 micrograms/dL    -   xi. Phosphorus—125.0 mg/dL to 225.0 mg/dL    -   xii. Potassium—190.0 mg/dL to 218.0 mg/dL    -   xiii. Sodium—50 mg/dL to 90 mg/dL    -   xiv. Zinc—2.0 mg/dL to 3.96 mg/dL

22. Once the nutritional analysis was complete, a label was generatedwith the actual values from the lab. Generally, a normal procedure forfoods is to put an average on the label. These Prolact products,however, show what was specifically in each bottle.

23. The product was shipped to customers in insulated coolers on dryice. Cool blocks can also be used in lieu of dry ice for shipping.

Example 2 Production and Compositions of Various Types of PROLACTA™Human Milk Products Based on Human Milk Fortifier

The human milk fortifiers (HMFs) produced, e.g., as described in Example1 supra, were mixed at various concentrations with raw human milk (e.g.,the milk from the mother of the premature infant). Depending on thenutritional content of mother's own milk and the needs of the infant,HMF can be mixed at various ratios. PROLACT+4™ is a composition meant tobe mixed at a ration of about 80% of raw milk with 20% of HMF.PROLACT+6™ is a composition meant to be mixed at a ratio of about 70%raw milk and 30% HMF. PROLACT+8™ is a composition meant to be mixed at aratio of about 60% raw milk and 40% HMF. PROLACT+10™ is meant to bemixed at a ratio of about 50% of raw milk and 50% of HMF. The mixing wasdone by the customers, not at the PROLACTA™ facilities. The table belowshows an exemplary nutritional comparison of nutrients in raw milk, andthe four PROLACTPLUS™ compositions mixed at various ratios with rawmilk.

TABLE 1 Nutritional Comparison of Raw Human Milk and Four PROLACTPLUSHuman Milk Fortifier Compositions Per 100 mL* Pre- term Nutrient UnitMilk+ PROLACT + 4 ™ PROLACT + 6 ™ PROLACT + 8 ™ PROLACT + 10 ™ mixingratios: 100% 80:20 70:30 60:40 50:50 mother to fortifier Volume mL 100100 100 100 100 Energy cal 68 84 91 99 107 protein g 1.3 2.2 2.7 3.2 3.7(human) carbohydrates g 7.0 7.6 7.9 8.2 8.5 fat (human)++ g 3.9 4.9 5.45.9 6.5 Minerals** Sodium mg 46 54 58 54 56 Potassium mg 66.3 71.1 73.571.1 72.3 Calcium mg 28 128 178 128 153 Phosphorus mg 14 63.1 87.7 63.175.4 Magnesium mg 3.3 8 10.4 8 9.2 Chloride mg 78.1 83.4 86.1 83.4 84.7Manganese mcg 0.4 2.36 3.2 2.36 2.79 Copper mg 0.08 0.1 0.1 0.1 0.1 Zincmg 0.5 0.7 1.1 0.7 0.9 Iron mg 0.1 0.2 0.2 0.2 0.2 Osmolality mOsm/ ≈290<335 <360 <325 <350 kgH₂O +calculated based on various published studieson file at Prolacta Bioscience ++data for pre-term milk is notavailable; fat is calculated based upon total calories *nutritionalvalues are based upon minimum values from Product ManufacturingSpecification for PROLACTPLUS ™ line of fortifiers **based upon averageof lots produced

Example 3 Production and Compositions of Standardized Human MilkFormulations

The PROLACTA™ Bioscience NEO20™ is a standardized human milk formulationthat was produced with the following steps. Processing was done in aclass 100,000 clean room (ISO class 8), and filling was done in a class10,000 clean room (ISO class 7):

1. Donor milk was pooled.

2. A sample was taken for Polymerase Chain Reaction (PCR) to test forthe following viruses:

a. HIV-1

b. HBV—Hepatitis B

c. HCV—Hepatitis C

3. Donor milk was filtered through 200 micron screen.

4. Whole milk underwent ultra filtration. This step filtered water outof the whole milk. The water was referred to as permeate. The finalconcentration of protein during this step was 1.2%-1.3%.

5. The filters were post-washed using permeate to recover any proteinthat was trapped on the filters during the ultra filtration process. Thepost wash was added to the concentrated whole milk until the proteinconcentration was 1.2%-1.5%.

6. During the Bulk formulation, cream from previous fortifier lots wasadded to the concentrated whole milk, after adding post wash to increasecalories to the correct target. At this point, the product was at:

a. Fat—3.5%-5.5%;

b. Protein—1.1%-1.3%; and

c. Lactose—8%-10.5%.

7. Next, the product was pasteurized in a tank. The tank was jacketedand hot glycol was used to heat up the tank. The following parameterswere followed:

a. The product temperature was 63° C. or greater;

b. The air temperature above the product was 66° C. or greater; and

c. The product was pasteurized for a minimum of 30 minutes.

8. The product temperature was brought down to 2° C.-8° C. using coldglycol in the jacketed tank.

9. The product was filled into bottles using a Watson-Marlow fillingmachine in the class 10,000 clean room. The fill sizes were as follows:

a. NEO20™ 30 mL;

b. NEO20™ 40 mL;

c. NEO20™ 50 mL;

d. NEO20™ 148 mL; and

e. NEO20™ 200 mL.

10. When the fill was finished, the bottles were taken for the followingtests:

a. BioBurden samples—2 bottles from the beginning, middle and end of thefill run were taken, for a total of 6 bottles;

b. Nutritional samples—1 bottle from the beginning, middle, and end ofthe fill run was taken, for a total of 3 bottles; and

c. Retention samples—120 mL of product was retained.

11. The following parameters were tested in the BioBurden samples:

a. Total Aerobic Count—<100 Colony Forming Unit/mL;

b. Bacillus cereus—<10 Colony Forming Unit/mL;

c. E. coli—<1 Colony Forming Unit/mL;

d. Coliform—<1 Colony Forming Unit/mL;

e. Pseudomonas—<1 Colony Forming Unit/mL;

f. Salmonella—<1 Colony Forming Unit/mL;

g. Staphylococcus—<1 Colony Forming Unit/mL;

h. Yeast—<100 Colony Forming Unit/mL; and

i. Mold—<100 Colony Forming Unit/mL.

12. Nutritional analysis was conducted for all fill sizes of NEO20™. Theconcentration values were in the following ranges:

i. Total Calories—0.69 Cal/mL to 0.74 Cal/mL;

ii. Protein—1.1 g/dL to 1.3 g/dL;

iii. Fat—3.5 g/dL to 5.5 g/dL; and

iv. Lactose—8.0 g/dL to 10.5 g/dL.

13. Once the nutritional analysis was complete, a label was generatedwith the actual values from the lab. Generally, a normal procedure forfoods is to put an average on the label. These PROLACTA™ products,however, show what was specifically in each bottle.

14. The product was shipped to customers in insulated coolers on dryice. Cool blocks can be used for shipping in lieu of dry ice.

Example 4 Production and Compositions of Standardized Human MilkFortified with Minerals

The PROLACTA™ Bioscience PROLACT20™ is a standardized human milk productfortified with minerals. It was produced using the following steps in aclass 100,000 clean room (ISO 8). Filling was done in a 10,000 cleanroom (ISO 7).

1. Donor milk was pooled.

2. A sample was taken for Polymerase Chain Reaction (PCR) tests for thefollowing viruses:

a. HIV-1

b. HBV—Hepatitis B

c. HCV—Hepatitis C

3. Donor milk was filtered through 200 micron screen.

4. Whole milk was ultra filtered. In this step water was filtered out ofthe whole milk. The water was referred to as permeate. The finalconcentration of protein during this step was 1.2%-1.3%.

5. The filters were post washed using permeate to recover any proteinthat was trapped on the filters during the ultra filtration process. Thepost wash was added to the concentrated whole milk until the protein wasin the range of 1.2%-1.5%.

6. During the Bulk formulation, cream from previous fortifier lots wasadded to the concentrated whole milk after adding post wash, to increasecalories to the correct target. At this point, the product was:

a. Fat—3.5%-5.5%;

b. Protein-1.1-1.3%; and

c. Lactose—8%-10.5%.

7. At this stage, the product can be frozen and later thawed for furtherprocessing.

8. Based on the analysis of the starting minerals, it was calculated howmuch more of minerals needed to be added. The final targets were:

a. Calcium—40 mg/dL to 150 mg/dL;

b. Chloride—30 mg/dL to 80 mg/dL;

c. Copper—0.05 mg/dL to 0.21 mg/dL;

d. Iron—0.1 mg/dL to 0.5 mg/dL;

e. Magnesium—3.0 mg/dL to 13 mg/dL;

f. Manganese—1.0 micrograms/dL to 9.2 micrograms/dL;

g. Phosphorus—15 mg/dL to 60 mg/dL;

h. Potassium—60 mg/dL to 120 mg/dL;

i. Sodium—20 mg/dL to 60 mg/dL; and

j. Zinc—0.25 mg/dL to 1.2 mg/dL.

9. After mineral addition (and thawing, if the product was frozen), theproduct was pasteurized in a tank. The tank was jacketed and hot glycolwas used to heat up the tank. The following parameters were followed:

a. The product temperature was 63° C. or greater;

b. The air temperature above the product was 66° C. or greater; and

c. The product was pasteurized for a minimum of 30 minutes.

10. The product temperature was brought down to 2° C.-8° C. using coldglycol in the jacketed tank.

11. The product was filled into bottles using a Watson-Marlow fillingmachine in the class 10,000 clean room. The fill sizes were as follows:

a. PROLACT20™ 30 mL

b. PROLACT20™ 40 mL

c. PROLACT20™ 50 mL

12. When the fill was done, the bottles were taken for the following:

a. BioBurden samples—2 bottles from the beginning, middle and end of thefill run were taken, for a total of 6 bottles;

b. Nutritional samples—1 bottle from the beginning, middle, and end ofthe fill run was taken, for a total of 3 bottles; and

c. Retention samples—120 mL of product was retained.

13. The following were tested in the BioBurden samples:

a. Total Aerobic Count—<100 Colony Forming Unit/mL;

b. Bacillus cereus—<10 Colony Forming Unit/mL;

c. E. coli—<1 Colony Forming Unit/mL;

d. Coliform—<1 Colony Forming Unit/mL;

e. Pseudomonas—<1 Colony Forming Unit/mL;

f. Salmonella—<1 Colony Forming Unit/mL;

g. Staphylococcus—<1 Colony Forming Unit/mL;

h. Yeast—<100 Colony Forming Unit/mL; and

i. Mold—<100 Colony Forming Unit/mL.

14. Nutritional analysis was conducted for all fill sizes of PROLACT20™after minerals were added. The ranges included:

i. Total Calories—0.69 Cal/mL to 0.74 Cal/mL;

ii. Protein—1.1 g/dL to 1.3 g/dL;

iii. Fat—3.5 g/dL to 5.5 g/dL;

iv. Lactose—8.0 g/dL to 10.5 g/dL;

v. Calcium—40 mg/dL to 150 mg/dL;

vi. Chloride—30 mg/dL to 80 mg/dL;

vii. Copper—0.05 mg/dL to 0.21 mg/dL;

viii. Iron—0.1 mg/dL to 0.5 mg/dL;

ix. Magnesium—3.0 mg/dL to 13 mg/dL;

x. Manganese—1.0 micrograms/dL to 9.2 micrograms/dL;

xi. Phosphorus—15 mg/dL to 60 mg/dL;

xii. Potassium—60 mg/dL to 120 mg/dL;

xiii. Sodium—20 mg/dL to 60 mg/dL; and

xiv. Zinc—0.25 mg/dL to 1.2 mg/dL.

15. Once the nutritional analysis was complete, a label was generatedwith the actual values from the lab. In general food procedures, anaverage is placed on the label. These PROLACTA™ products, however, showwhat was specifically in each bottle.

16. The product was shipped to customers in insulated coolers on dryice. Cool blocks can also be used for shipping in lieu of dry ice.

Example 5 Validation of the Pasteurization Methods

The methods and compositions of the disclosure maintain desirableactivities of important proteins and vitamins in the describedcompositions and eliminate bioburden (see, e.g., Terpstra at al.,Breastfeeding Med. 2(1):27-33, 2007).

A. Bioburden Validation

A validation of the effects of high-temperature short-time (HTST)pasteurization and vat pasteurization on the bioburden of human milk wascarried out. The test organisms used in the validation studies were thefollowing bacteria and viruses: E. coli, S. aureus, and S. agalactiae,human immunodeficiency virus (HIV), hepatitis A virus (HAV), bovineviral diarrhea virus (BVDV) and pseudorabies virus (PSR). HIV and HAVare known to be potential contaminants of human milk and were,therefore, selected as relevant viruses. Hepatitis C virus (HCV) is alsoknown to be a potential contaminant of human milk. However, this viruscannot be cultured effectively in laboratory cell line systems so thespecific model virus BVDV is used. For the same technical reason,hepatitis B virus (HBV), also a known potential contaminant of humanmilk, is substituted by the general model pseudorabies virus which, likeHBV, is a lipid-enveloped virus. Using the methods described herein, thefollowing results were obtained:

TABLE 2 Log₁₀ Reduction Values for Test Organisms Log₁₀ Pathogen Log₁₀Reduction (HTST) Reduction (vat) E. coli >32 Not done S. aureus 15 Notdone S. agalactiae >26 Not done BVDVa >5.84 >6.1 HIV >7.27 >6.7PSR >7.68 >6.8 HAV ~2 ~1.7

These viral log reduction values do not represent the maximum reductionthat can be achieved by the methods of the disclosure. Although S.aureus is fairly resistant to heat treatment, this organism showed a 15log reduction with the process of the disclosure.

B. Validation of Milk Components

A validation of the level of various human milk components afterpasteurization was also carried out.

Immunoglobulin A (IgA) and secretory IgA (s[IgA₂]) were quantitated inhuman milk samples using sandwich ELISA procedures. Followingpasteurization using the HTST process featured herein, IgA concentrationdeclined about 27% (e.g., a range of about 7% to about 47%), on average,and secretory IgA levels declined 17% (e.g., a range of about 7% toabout 27%), on average, compared to the corresponding values inuntreated human milk samples.

Lysozyme activity was determined by a microtiter assay using aMicrococcus lysodeikticus suspension as a substrate. The lysozymeactivity in human milk after pasteurization was about 22,000 IU/mL, 57%(e.g., a range of about 47% to about 67% or more) of the initialactivity (39,000 IU/mL) in raw human milk.

Lactoferrin concentration was determined by an ELISA technique. Thelactoferrin content of human milk after pasteurization using the methodsof the disclosure was about 0.033 g/100 mL, about 14% (e.g., a range ofabout 4-24%) of the initial concentration (0.24 g/100 mL) in raw humanmilk.

Vitamin analyses were performed by validated HPLC procedures. Vitamin A,vitamin C, and α-, γ-, and δ-tocopherol levels remained unchangedfollowing pasteurization. The vitamin B6 content of human milk slightlydecreased to about 7.8 μg/100 mL, about 89% of the initial concentrationof 8.8 μg/100 mL. These results are presented in Table 3.

TABLE 3 Effect of Pasteurization on Human Milk Constituents UntreatedPasteurized % Constituent Milk Milk Remaining Immunoglobulin A (mg/mL)315 230 73 Secretory IgA (mg/mL) 462 379 82 Lysozyme (IU/mL) 39,00022,000 57 Lactoferrin (g/100 mL) 0.24 0.033 14 Vitamin B6 (μg/100 mL)8.8 7.8 89

Other variations and embodiments of the invention described herein willnow be apparent to those of ordinary skill in the art without departingfrom the scope of the invention or the spirit of the claims below.

1-65. (canceled)
 66. A method for obtaining a pasteurized human milkfortifier, the method comprising: (a) genetically screening pooled humanmilk for one or more viruses selected from the group consisting ofHIV-1, HBV and HCV; (b) filtering the pooled human milk; (c)heat-treating the pooled human milk: (d) separating the pooled humanmilk into cream and skim; (e) adding a portion of the cream to skim suchthat the resulting composition comprises: (i) 35-85 mg/mL of protein(ii) 60-110 mg/mL of fat (iii) 60-140 mg/ml, of carbohydrate; and (f)pasteurizing the resulting composition from (e) thereby obtaining apasteurized human milk fortifier.
 67. The method of claim 66, whereinthe heat-treating comprises treating the pooled human milk at about 63°C. or greater for about 30 min.
 68. The method of claim 66, furthercomprising ultrafiltration of the skim after step (d).
 69. The method ofclaim 66, wherein the genetic screening in step (a) comprises utilizingthe polymerase chain reaction.
 70. The method of claim 66, wherein thepooled human milk is filtered through a 200 micron filter in step (b).71. The method of claim 66, further comprising running the cream througha separator following step (d).
 72. The method of claim 68, whereinultrafiltration the skim comprises filtering permeate out of the skim.73. The method of claim 68, further comprising washing the ultrafiltersto obtain post wash solution.
 74. The method of claim 73, furthercomprising combining the post wash solution to skim.
 75. The method ofclaim 66, further comprising carrying out mineral analysis of thecomposition obtained after step (e).
 76. The method of claim 66, furthercomprising adding to the resulting composition obtained after step (e)one or more minerals selected from the group consisting of calcium,chloride, copper, iron, magnesium, manganese, phosphorus, potassium,sodium, and zinc.
 77. The method of claim 66, further comprisingcarrying out biological testing of a portion of the pasteurized humanmilk fortifier after step (f).
 78. The method of claim 66, furthercomprising carrying out nutritional testing of a portion of thecomposition after step (f).
 79. The method of claim 66 wherein adding aportion of the cream to skim in step (e) is done such that the resultingcomposition comprises: (i) 55-65 mg/mL of protein (ii) 60-110 mg/mL offat (iii) 60-140 mg/mL of carbohydrate.
 80. A method for obtaining apasteurized human milk fortifier, the method comprising: (a) geneticallyscreening pooled human milk by polymerase chain reaction for one or moreviruses selected from the group consisting of HIV-1, HBV and HCV; (b)filtering the pooled human milk through a 200 micron screen; (c)heat-treating the pooled human milk at about 63° C. or greater for about30 minutes; (d) separating the pooled human milk into cream and skim;(e) ultrafiltering the skim obtained in step (d) to remove permeate (f)adding a portion of the cream obtained in step (d) to the ultrafilteringskim obtained in step (e) such that the resulting composition comprises:(i) 55-85 mg/mL of protein (ii) 60-110 mg/mL of fat (iii) 60-140 mg/mLof carbohydrate; (g) carrying out mineral analysis on the composition;(h) adding one or more of the minerals to the composition wherein theone or more minerals are selected from the group consisting of: calcium,chloride, copper, iron, magnesium, manganese, phosphorus, potassium,sodium, and zinc; and (i) pasteurizing the resulting composition from(h) thereby obtaining at pasteurizing human milk fortifier.